Summary Statement. A porcine model using Yucatan minipigs was found to be very promising for the investigation of healing around transcutaneous osseointegrated implants. Pigs demonstrated surprising agility and adaptability including the ability to ambulate on three legs during the immediate postoperative period. Introduction. Previous non weight-bearing and weight-bearing caprine, canine and ovine models have evaluated design, material, and biological coating variations in an attempt to improve the wound healing and skin-implant seal around transcutaneous osseointegrated implants. Although these models have primarily been used as a window into the application of transcutaneous osseointegrated implants in humans, some important model characteristics affecting wound healing and infection have been missing including: 1) replication of the physiological tissue response, and 2) availability of a transcutaneous site with sufficient soft tissue coverage. Pig skin, like human, is relatively hairless, tightly attached to the subcutaneous tissue, vascularised by a cutaneous blood supply, and healed by means of epithelialization. Swine have been extensively utilised for superficial and deep wound healing studies and can offer ample soft tissue coverage following a lower limb amputation. Development of a porcine model is important for continued understanding and improvement of weight-bearing transcutaneous osseointegration. Methods. Two male Yucatan mini-pigs (9 months, 36kg) were fit with transcutaneous osseointegrated prostheses using a single-stage transtibial amputation and prosthesis implantation procedure. The endo-prosthesis consisted of a cylindrical intraosseous threaded section and a smooth transcutaneous section. The transcutaneous sections were smooth to promote epithelialization and deter direct skin-implant adhesion. The implants were custom manufactured from medical grade Ti-6Al-4V alloy. The exo-prosthesis, consisting of an adjustable length leg and foot, was attached by clamp to the supercutaneous portion of the implant following either one or two days of sling constraint to limit initial weight-bearing. Various exo-prosthesis designs and configurations were trialed. The animals’ behavior and gait were closely observed. Weight-bearing was monitored using a force plate. At 5 and 8 weeks, clinical, microbiological, and histological data were examined to assess wound healing and infection at the skin-bone-implant interface. Results. The pigs demonstrated surprising agility and adaptability. They were able to successfully ambulate on three legs during the post-op period before weight-bearing was permitted. They adapted quickly to changes in exo-prosthesis design, position, and length. Although bacterial colonization was verified, neither of the animals exhibited clinical signs of infection over the respective eight and five week studies. Histological results indicated that there was no skin to implant adhesion but that epithelial growth was progressing towards the implant in one animal. Healing of the transcutaneous wound site showed substantial progress but a definitive skin seal was non-existent at the eight week time point. Discussion/Conclusion. This is likely the first animal model developed, having soft tissue characteristics similar to those found in humans, in which an axially-loaded, weight-bearing implant was successfully used. Results indicated that this porcine model offers many advantages over previous models for the development, evaluation, and comparison of the various techniques being advocated to achieve successful transcutaneous osseointegration in humans. The Yucatan miniature pig's ability to ambulate on three legs during the immediate post-operative period and quickly adjust to changes in the exo-prosthesis design, coupled with their physiological similarity to humans, makes them a valuable model for future studies

Conventional amputation prostheses rely on the attachment of the socket to the stump, which may lead to soft-tissue complications. Intraosseous transcutaneous amputation prostheses (ITAPs) allow direct loading of the skeleton, but their success is limited by infection resulting from breaching of the skin at the interface with the implant. Keratinocytes provide the skin’s primary barrier function, while hemidesmosomes mediate their attachment to natural ITAP analogues. Keratinocytes must attach directly to the surface of the implant. We have assessed the proliferation, morphology and attachment of keratinocytes to four titaniumalloy surfaces in order to determine the optimal topography in vitro. We used immunolocalisation of adhesion complex components, scanning electron microscopy and transmission electron microscopy to assess cell parameters. We have shown that the proliferation, morphology and attachment of keratinocytes are affected by the surface topography of the biomaterials used to support their growth. Smoother surfaces improved adhesion. We postulate that a smooth topography at the point of epithelium-ITAP contact could increase attachment in vivo, producing an effective barrier of infection

Intraosseous Transcutaneous Amputation Prosthesis (ITAP) is a new generation of limb replacements that can provide to amputees, an alternative solution to the main problems caused by the most common used external prosthesis such as pressure sores, infections and unnatural gait. ITAP is designed as one pylon osteointegrated into the bone and protruding through the skin, allowing both the mechanical forces to be directly transferred to the skeleton and the external skin being free from frictions and infections. The skin attachment to the implant is fundamental for the success of the ITAP, as it prevents the implant to move and consequently fail. In this study we wanted to test if cell viability and attachment was improved using TiO2 nanotubes. Human keratinocytes and human dermal fibroblasts were seeded for three days on TiO2 nanotubes with different sizes (18–30nm, 40–60nm and 60–110nm), compared with controls (smooth titanium) and tested for viability and attachment. A Mann-Whitney U test was used to compare groups where p values < 0.05 were considered significant. The results showed that the viability and cell attachment for keratinocytes were significantly higher after three days on controls comparing with all nanotubes (p=0.02), while attachment was higher on bigger nanotubes and controls. Cell viability for fibroblasts was significantly higher on nanotubes between 40 and 110nm comparing with smaller size and controls (p=0.03), while investigation of cell attachment is ongoing. From these early results, we can say that TiO2 nanotubes can improve the soft tissue attachment on ITAP. Further in-vitro and ex-vivo experiments on cell attachment will be carried out

Introduction and Objective. Knee osteoarthritis (KOA) is a frequent disease for which therapeutic possibilities are limited. In current recommendations, the first-line analgesic is acetaminophen. However, low efficacy of acetaminophen, frequently leads to the use of weak opioids (WO) despite their poor tolerance, especially in elderly patients. The primary objective was to compare the analgesic efficacy and safety of a new wearable transcutaneous electrical nerve stimulation (W-TENS) to weak opioids (WO) in the treatment of moderate to severe, nociceptive, chronic pain in knee osteoarthritis patients. Materials and Methods. ArthroTENS study is a phase 3, non-inferiority, multicentric, prospective, randomized, single-blinded for primary efficacy outcome, controlled, in 2-parallel groups, clinical study comparing W-TENS versus WO over a 3-month controlled period with an additional, optional, non-controlled, 3-month follow-up for patients in W-TENS group. The co-primary outcome was KOA pain intensity (PI) at month 3 and the number of adverse events (AEs) over 3 months. Results. The non-inferiority of W-TENS was demonstrated in both the PP and ITT populations. At M3, PI in PP population was 3.87 (2.12) compared to 4.66 (2.37) (delta: −0.79 (0.44); 95% CI (−1.65; 0.08)) in W-TENS and WO groups, respectively. Since the absolute value of the 95% CI of the between-treatments mean PI difference [−1.71, – 0.12] was above 0 in ITT set, the planned superiority analysis was performed, demonstrating that W-TENS was significantly superior to WO at M3 (P=0.0124). At M1 and M3, the W-TENS group reached the absolute minimal clinically important difference (MCID) for an analgesic (1.8 (2.1) and 2.1 (2.3), respectively), corresponding to a 20 mm reduction in PI (interquartile range: 15–30) on a 0–100 mm visual analogic scale – i.e. 2 points on a numerical rating scale – which equates to “much better”. Conversely, in the WO group, a 0.5 (1.8) and a 1.1 (2.1) reduction in PI were observed at M1 and M3, respectively, while a 1-point reduction in PI is required to be considered as a “slightly better” improvement. In WO group, AEs were the common systemic AEs reported with WO (nausea, constipation, drowsiness, dizziness, pruritus, vomiting, dry mouth). AEs in W-TENS group were local, such as local cutaneous reaction (erythema). Thirty-nine (70.9%) patients wished to extend W-TENS treatment for 3 additional months. Only one patient discontinued this additional period and results were maintained at M6. Conclusions. W-TENS was more effective and better tolerated than WO in the treatment of nociceptive KOA chronic pain and could represent an interesting non-pharmacological alternative to WO

Infection is the primary failure modality for transcutaneous implants because the skin breach provides a route for pathogens to enter the body. Intraosseous transcutaneous amputation prostheses (ITAP) are being developed to overcome this problem by creating a seal at the skin-implant interface to prevent bacterial invasion. Oral gingival epithelial cell adhesion creates an infection free seal around dental implants; however this has yet to be demonstrated outside the oral environment. All epithelial cells attach via hemidesmosomes (HD) and focal adhesions (FA) and their expression is an indicator of adhesion efficiency. The aim of this study was to compare epidermal keratinocyte with oral gingival epithelial cell adhesion on titanium alloy in vitro to determine whether these two cell types differ in their speed and strength of adhesion. It was hypothesised that oral gingival epithelial cells attach to titanium alloy earlier than epidermal keratinocytes; with greater expression of hemidesmosomes and focal adhesions. Human oral gingival epithelial cell (HGEP) and primary human epidermal keratinocyte (HPEK) adhesion to titanium alloy, was assessed at 4, 24, 48 and 72 hrs. Adhesion was measured by the number of FAs per unit cell area and expression of HDs using a semi-quantitative scale. At 4 and 24hrs, there was a significant increase in vinculin marker expression per unit cell area of 4.3 and 4.7 times in HGEP compared with HPEK (p=0.000). At 48 and 72hrs there were no significant differences. HD expression was significantly greater in HGEP at 4 and 24hrs (p=0.002) compared with HPEK. Up-regulation of HD expression in HPEK lagged that of HGEP until 48hrs, after which no significant differences were observed. This study has demonstrated that oral gingival cells up-regulate both focal adhesion and hemidesmosome expression at earlier time points compared with epidermal keratinocytes. Expression of hemidesmosomes lags that of focal adhesions, suggesting that focal adhesion formation is a prerequisite for hemidesmosome assembly. We postulate that early attachment of oral gingival epithelial cells to dental implant biomaterials may be responsible for the formation of an infection-free seal

To try and aid the formation of a soft tissue seal to promote dermal and epidermal attachment to Intraosseous Transcutaneous Amputation Prostheses we compared the effect of titanium surfaces functionalised with fibronectin (fn) or YRGD peptide sequences on human dermal cell (HDF) attachment. We hypothesise that YRGD and fn coatings will significantly increase HDF attachment to titanium alloy substrates. Titanium alloy 10mm discs were polished and acted as control substrates, functionalised surfaces had YRGD or fn adsorbed or silanised onto the polished surface. HDFs were seeded at 10,000/disc and cultured for 1, 4, 24 and 96 hours, fixed and fluorescent immnolocalisation for vinculin was performed. Individual vinculin markers were counted and density calculated as a measure of cell attachment. All assays were performed in triplicate and data were analysed in SPSS 19.0 and results were considered significant at the 0.05 level. Results showed an up-regulation of Focal adhesion density (FA) against controls at all time-points (excluding ad-fn at 4 hours, p=0.057), p values < 0.05, the use of functionalised titanium surfaces may lead to long-term clinical success of ITAP. We have shown a significant positive effect on cell attachment when a synthetic peptide sequence is used. Using synthetic peptide sequence may also be more beneficial from a regulatory stand-point compared with using isolated proteins

The success of long-term transcutaneous implants depends on dermal attachment to prevent downgrowth of the epithelium and infection. Hydroxyapatite (HA) coatings and fibronectin (Fn) have independently been shown to regulate fibroblast activity and improve attachment. In an attempt to enhance this phenomenon we adsorbed Fn onto HA-coated substrates. Our study was designed to test the hypothesis that adsorption of Fn onto HA produces a surface that will increase the attachment of dermal fibroblasts better than HA alone or titanium alloy controls. . Iodinated Fn was used to investigate the durability of the protein coating and a bioassay using human dermal fibroblasts was performed to assess the effects of the coating on cell attachment. Cell attachment data were compared with those for HA alone and titanium alloy controls at one, four and 24 hours. Protein attachment peaked within one hour of incubation and the maximum binding efficiency was achieved with an initial droplet of 1000 ng. We showed that after 24 hours one-fifth of the initial Fn coating remained on the substrates, and this resulted in a significant, three-, four-, and sevenfold increase in dermal fibroblast attachment strength compared to uncoated controls at one, four and 24 hours, respectively

According to the latest report from the German Arthroplasty Registry, aseptic loosening is the primary cause of implant failure following primary hip arthroplasty. Osteolysis of the proximal femur due to the stress-shielding of the bone by the implant causes loss of fixation of the proximal femoral stem, while the distal stem remains fixed. Removing a fixed stem is a challenging process. Current removal methods rely on manual tools such as chisels, burrs, osteotomes, drills and mills, which pose the risk of bone fracture and cortical perforation. Others such as ultrasound and laser, generate temperatures that could cause thermal injury to the surrounding tissues and bone. It is crucial to develop techniques that preserve the host bone, as its quality after implant removal affects the outcome of a revision surgery. A gentler removal method based on the transcutaneous heating of the implant by induction is proposed. By reaching the glass transition temperature (T. G. ) of the periprosthetic cement, the cement is expected to soften, enabling the implant to be gently pulled out. The in-vivo environment comprises body fluids and elevated temperatures, which deteriorate the inherent mechanical properties of bone cement, including its T. G. We aimed to investigate the effect of fluid absorption on the T. G. (ASTM E2716-09) and Vicat softening temperature (VST) (ISO 306) of Palacos R cement (Heraeus Medical GmbH) when dry and after storage in Ringer's solution for up to 8 weeks. Samples stored in Ringer's solution exhibited lower T. G. and VST than those stored in air. After 8 weeks, the T. G. decreased from 95.2°C to 81.5°C in the Ringer's group, while the VST decreased from 104.4°C to 91.9°C. These findings will be useful in the ultimate goal of this project which is to design an induction-based system for implant removal. Acknowledgements: Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB/TRR-298-SIIRI – Project-ID 426335750

Summary Statement. A novel transcutaneous CO. 2. therapy significantly enhanced the antitumor effectiveness of X-ray irradiation in human MFH xenografts The results strongly suggest that transcutaneous CO. 2. therapy could be a novel therapeutic tool for overcoming radioresistance in human malignancies. Introduction. Hypoxia contributes to tumor radioresistance. In the presence of oxygen, reactive oxygen species (ROS) play crucial roles in cellular apoptosis to irradiation. We previously showed that a novel transcutaneous application of CO. 2. can improve hypoxia and that it induces apoptosis and decreases the expression of HIF-1α in sarcoma. Therefore, we hypothesised that a transcutaneous application of CO. 2. may increase radiosensitivity in sarcoma by improvement of hypoxic condition and increasing ROS production in tumors. The purpose of this study is to examine the effect of transcutaneous application of CO. 2. on radiosensitivity in human malignant fibrous histiocytoma (MFH) cells. Methods. Cells. We used a human MFH cell line, Nara-H in this study. X-ray irradiation. X-ray irradiation was performed at a dose rate of 0.64 to 0.66 Gy/min. Colony formation assay. In vitro cell viability after X-ray irradiation was assessed by colony formation assay. In vivo studies. Nara-H cells were subcutaneously implanted to 24 nude mice which were randomly divided into 4 groups; CO. 2. group, X-ray group, Combination group and Control group. CO. 2. therapy was performed as we previously reported (1, 2). In combination group, mice were treated twice a week by X-ray at 3.2 Gy shortly after CO. 2. therapy for 2 weeks. The changes in body weight and tumor volume were monitored for 14 days in all 4 groups. The implanted tumors were excised at the end of experiment. We also excised tumors on the first day of each treatment in all 4 groups, and examined apoptosis and ROS expression by FACS analysis. The animal experiments were approved by the Animal Committee in our institute. Immunoblot analysis. The protein expression of HIF-1α, ROS-related proteins (p38 and JNK/SAPK), and apoptosis-related proteins (caspase-3 and PARP) were assessed by immunoblot analysis. FACS analysis. DNA fragmentation and ROS production in tumors were assessed by FACS analysis. Statistical analysis. ANOVA with post hoc test to compare for continuous values. All tests were considered significant at p<0.05. Results. Approximately 50% of Nara-H cells survived after a total of 3.2 Gy X-ray irradiation. Tumor volume in combination group was significantly reduced at the end of experiment (47% of that in X-ray group and 28% of that in control group). In Combination group, apoptosis with ROS production markedly increased when compared with those in Control, CO. 2. or X-ray group at 24 hours after treatment. Immunoblot analysis showed that, in combination group, the expression of phospho-p38, phospho-JNK/SPAK, and cleavage of both caspase-3 and PARP were increased compared with other groups, conversely, the expression of HIF-1α was decreased. Discussion/Conclusion. In this study, we demonstrated that the combination therapy showed more significant effects on apoptosis and ROS production through improving hypoxia in MFH cells in vivo. Our findings strongly suggest that the combination therapy of CO. 2. and X-ray could be a novel therapeutic strategy for the treatment of human MFH, and that transcutaneous application of CO. 2. may be one of the best radiosensitisers

Summary. Osseointegrated Amputation Prostheses can be functionalised by both biological augmentation and structural augmentation. These augmentation techniques may aid the formation of a stable skin-implant interface. Introduction. Current clinical options are limited in restoring function to amputees, and are associated with contact dermatitis and infection at the stump-socket interface. Osseointegrated Amputation Prosthesis attempts to solve issues at the stump-socket interface by directly transferring axial load to the prosthesis, via a skin-penetrating abutment. However, development is needed to achieve a seal at the skin-implant interface to limit infection. Fibronectin, an Extracellular Matrix protein, binds to integrins during wound healing, with the RGD tripeptide being part of the recognition sequence for its integrin binding domain. In vitro work has found silanization of RGD to polished titanium discs up regulates fibroblast attachment compared to polished control. Electron Beam Melting can produce porous titanium alloy implants, which may encourage tissue attachment. This study aims to test whether a combination of biological RGD coatings and porous metal manufacturing techniques can encourage the formation of a seal at the skin-implant interface. Materials and Methods. We developed four different augmented transcutaneous devices: Porous, Porous RGD coated, drilled and drilled RGD coated. These were implanted in tibial transcutaneous ovine model, n=6, for a period of 6 months. Following explantation we performed hard grade resin histology to assess soft tissue attachment at the transcutaneous interface. Results. Histological analysis revealed no statistical difference in epithelial downgrowth and epidermal attachment values between the four augmented devices. There were significant increases (p<0.05) in the number of blood vessels and the number of cells in the Porous RGD devices compared with both drilled implant devices. Both Porous and Porous RGD implant groups observed significant increase (p<0.05) in soft tissue infiltration compared with both Drilled implant devices. Discussion. The use of porous structures and RGD coatings increases tissue ingrowth and revascularisation in ITAP devices despite having no effect on epithelial downgrowth and epidermal attachment in a long-term ovine model. There were no detrimental effects in the transcutaneous interface formation observed. These augmentation techniques may prove beneficial in preclinical and clinical developments of transcutaneous osseointegrated devices

Development of more effective diagnostic and therapeutic solutions is vital to tackling the growing challenge of bone diseases and disorders in aging societies. Spatially offset Raman spectroscopy (SORS) enables the chemical specificity of conventional Raman spectroscopy to be combined with sub-surface probing. SORS has successfully been applied to transcutaneous investigations of underlying bone and shows great potential to become an in vivo tool for non-invasive diagnosis of various bone conditions. The volume within the complex hierarchical bone tissue probed by SORS depends on the specimen's optical properties. Understanding the actual sampling depth is important to correctly assign detected chemical changes to specific areas in the bone. This study explores the hypothesis that the effective Raman signal recovery from certain depths requires different spatial offsets depending on the bone mineralisation. SORS depth investigations were conducted on three bones with significantly different mineralisation levels. Thin slices (0.6 – 1.0 mm thickness) were cut from deer antler, horse metacarpal and whale tympanic bulla and stacked together (4 – 7 layers; 4.1 – 4.7 mm total thickness). A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as reference sample and was inserted in between the layers of stacked bone slices. Raman spectra were acquired at 30 s using 830 nm excitation. A quantitative relation between the SORS offset and the primarily interrogated depth inside the bone was established. Maximum accessible depths at small offset strongly depend on the mineralisation level. Using large spatial offsets of 7 – 9 mm PTFE signal recovery depths of 4.4 – 4.6 mm through cortical bone can be realized with only minor dependence on the bone mineralisation. These findings highlight the potential of SORS for medical diagnostics by enabling the non-invasive detection of bone conditions characterised by chemical alterations several millimetres inside compact bone tissue (e.g. infections, tumours, etc.)

Introduction. The interstitial fluid of bone fluid flow is supplied by flowing blood. Blood flow is determined by three kinds of muscles: cardiac, smooth, and skeletal. Cardiac muscle establishes baseline blood pressure. Smooth muscle controls vessel diameter and skeletal muscle creates intermittent intravascular pressure pulses. For the tibia the relevant skeletal muscle is the gastrocnemius which functions as a muscle pump. This study tested the hypotheses: 1) skeletal muscle-caused pressure pulses increase cortical blood flow, 2) extravasation of vascular fluid and, consequently, interstitial bone fluid flow are enhanced by resultant increased microvascular pressure and 3) bone healing is enhanced by increased bone fluid flow. Methods. Eighteen skeletally mature female New Zealand white rabbits were implanted with bone chamber windows (BCIs) as described previously. The windows were exposed at three weeks and observed weekly until Week 10 using intravital microscopy. During observation, the subject was suspended in prone position in a mesh fabric torso sling jacket so as to eliminate gravity-based reaction forces. Electrodes of a transcutaneous electrical nerve stimulator (TENS) were gel-glued at each rabbits gastroc-soleus position; but activated only in the 11 experimentals. A 4Hz 2.8 ± 1.3V impulse was delivered for 60 minutes. Still and video images were obtained at 0, 2, and 60 minutes following injection of 1μm fluorescent microspheres. Each such injection was followed by injection of 70 kD FITC- or RITC-dextran to define vascularity and capillary filtration. Additional still images were obtained at 5, 30, and 55 minutes. Muscle contraction forces during TENS were obtained acutely following the Week 10 observation with a Futek force transducer cell through an attached nylon suture. Bone mineral density was obtained at Week 3 and Week 10 with a Stratec pQCT and associated software. Data were analyzed statistically using a Wilcoxon signed rank test. Results. All three hypotheses were supported statistically by the data. The average force produced by TENS stimulated gastrocnemius contraction was 18.98 ± 9.42 N/kg muscle. This produced a microstrain of 192μe in bone around the BCI. Bloodflow results are shown in the figure. On average, flow decreased in controls by 12.6% and increased in experimentals by about 2%. Capillary filtration in experimentals was about 34.6% higher than controls after 60 minutes of TENS. Bone formation rate was 62.5% higher with TENS. Conclusion. In order to understand the role of fluid flows in bone physiology, we need to know the how and where of movement. These results suggest the part played by skeletal muscle in bone fluid movement cannot be ignored. As with many evolutionary adaptations, the muscle pump's hydrodynamic contribution to bone may be redundant and merely serve as a backup to percolation from poroelastic deformation. On the other hand, it may be crucial in disuse osteoporosis instigating conditions such as microgravity. The measured increases in capillary filtration and blood flow suggest that intravascular pressure which drives the former and resultant percolation has been increased by the muscle pump. It follows that fluid shear on cortical bone cells also increased. The challenge now is to obtain local flow measurements that would tell us how much

Objectives

Infection of implants is a major problem in elective and trauma surgery. Heating is an effective way to reduce the bacterial load in food preparation, and studies on hyperthermia treatment for cancer have shown that it is possible to heat metal objects with pulsed electromagnetic fields selectively (PEMF), also known as induction heating. We therefore set out to answer the following research question: is non-contact induction heating of metallic implants effective in reducing bacterial load in vitro?

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